molly ve lepıstes

Synaptonemal complex analysis of interspecific hybrids of Poecilia (Teleostei, Poecilidae).

ABSTRACT.

The pattern of chromosome pairing at meiotic prophase in males of the guppy (Poecilia reticulatus), black molly (Poecilia sphenops) marble molly (Poecilia velifera) and hybrids between these species was examined by means of electron microscopy of surface spread synaptonemal complexes. Meiotic chromosomes of the pure species and P. sphenops x P. velifera hybrid showed complete pairing at pachytene. No indication of heteromorphism for any chromosomes was found. The vast majority of pachytene cells of P. velifera x P. reticulatus hybrid demonstrated various signs of pairing failure: univalents, interlocks, multiple non-homologous pairing and end-to-end associations. However, a few cells were found to contain all completely paired homomorphic bivalents. These findings imply that: (1) genetic divergence between these species was not accompanied by gross chromosomal rearrangements; (2) pairing failure in the P. velifera x P. reticulatus hybrid is apparently determined by genic difference of the species-specific mechanism controlling meiotic prophase, rather than chromosome divergence.

INTRODUCTION

The comparative analysis of the karyotypes of closely related species has helped in understanding of the mechanisms of chromosomal evolution and phylogenetic relationships in animals, especially in mammals. The study of the chromosome evolution of fishes is inherently difficult because their chromosomes are usually not accessible for G-band staining (Sola et al., 1981). The similarity in morphology of routine stained chromosomes does not necessarily reflect a true homology between them. The resolution of classical cytogenetic methods is insufficient to detect chromosome rearrangements other than those changing chromosome number and/or arm ratio.

An alternative way to estimate the degree of homology between the chromosomes of closely related species is to observe their pairing in the meiotic prophase of inter-species hybrids (Liming and Pathak, 1981; Poorman, 1982; Derr et al., 1991; Dollin et al., 1991; Hale et al., 1993). A wide variety of chromosome rearrangements may be detected by means of electron microscopic analysis of the surface spread silver stained synaptonemal complexes (SC). Heterozygosity for the rearrangements (as well as heteromorphism for the sex chromosomes) displays at the level of SC as heteromorphic synaptic configurations (Moses, 1980).

The aim of this study was to examine the pattern of chromosome pairing at meiotic prophase in males of the guppy (Poecilia reticulatus), black molly (Poecilia sphenops), marble molly (Poecilia velifera) and the hybrids between them.

Karyotypes of P. reticulatus (Yosida and Hayashi, 1970; Scheel, 1972, Nanda et al., 1990), P. sphenops (Prehn and Rasch, 1969, Rishi and Gaur 1976, Haaf and Schmid, 1984), and P. velifera (Post, 1965; Prehn and Rasch, 1969) have already been described. All of them contain 23 pairs of acrocentric chromosomes. Data of Post (1965) suggesting n = 24 for P. sphenops have not been confirmed.

Genetic data indicate that P. reticulatus has an XX/XY system of sex determination (see Kirpichnikov, 1981 for review). Nanda et al. (1990, 1992) reported a difference in the C-band patterns between the telomeres of the homologous No. 1 chromosomes in male. One of homologues (Y) contained more telomeric heterochromatin that the other (X). Nanda et al. (1990, 1992) demonstrated that this difference was determined by accumulation of simple repeats on the telomeric heterochromatin of the Y chromosome. A distinct C-heteromorphism between two homologous chromosome 1 was observed in P. sphenops females (Haaf and Schmid, 1984), and interpreted as indication to the WZ/ZZ system of sex determination. Such heteromorphism has not been found in P. velifera indicating that the Z and W are still structurally equal (Nanda et al. 1992).

MATERIAL AND METHODS

Three adult males of each of the three species: P. reticulatus, P. sphenops, P. velifera, six of P. sphenops x P. velifera and two of P. velifera x P. reticulatus hybrids were used in this study. Female P. sphenops were crossed to male P. velifera and female P. velifera to male P. reticulatus, to generate interspecific hybrids.

The technique for obtaining microspread spermatocytes for analysis of the whole cell complements of SC was essentially the same as that of Speed (1982). Testes were removed and placed in Hanks medium in watchglasses. Then they were transferred onto a separate slide with three drops of 0.15 M sucrose. The testes were torn apart to free the germ cells. The cell suspension was transferred onto a slide precoated with 0.5% Optilux plastic in chloroform. The slides were dried and fixed with 4% paraformaldehyde in O.1M sucrose for 1O min., washed, dried and stained with silver nitrate. The spreads of good quality after light microscopic examination were transferred to specimen grids, and examined and photographed with an electron microscope JEM-100 (JEOL, Japan) at 80kV.

Approximately 100 spermatocytes per specimen were analysed in P. reticulatus, P. sphenops, P. velifera and P. sphenops x P. velifera , and 869 cells in P. velifera x P. reticulatus hybrids.

RESULTS AND DISCUSSION

Pure species.

In general, meiotic prophase in spermatocytes of P. reticulatus, P. sphenops and P. velifera has a similar appearance and was similar to that described for other fish (Lin and Yu, 1991). The formation of SC started at zygotene and completed at pachytene. A very small portion of zygotene cells was found. This indicates that zygotene is a rather short stage in these species. Pairing was mainly initiated at the chromosome ends, but some interstitial sites of synaptic initiation were also detected.

At pachytene, 23 completely paired bivalents were found in the males of all three species studied. It was impossible to identify individual SCs in any species as they formed a continuous series when arranged in decreasing size.

Taking into account the data of Nanda et al. (1990, 1992) about the C-band heteromorphism in chromosome 1 in males of P. reticulatus, we paid a special attention to the longest bivalent in SC complement of male guppies. We found that its lateral elements had equal length, were uniformly stained and completely paired with each other in the majority of pachytene cells. No one bivalent had the axes of unequal length. Some bivalents displayed a temporary asynapsis of the terminal segments at early and late pachytene, but usually there were several of these.

This means that the terminal regions of the X and Y chromosomes of the guppy pair non-homologously with each other. Non-homologous synapsis in structural heterozygotes has been found in many species. It has been shown that in male mammals the Y chromosome undergoes extensive non-homologous pairing with the differentiating part of the X chromosome (Ashley, 1987, Chandley et al. 1984; Pack et al. 1993; Tres, 1977). The lateral elements of Z and W chromosomes in birds demonstrate an ability to equalize their size in heteromorphic bivalents. (Solari, 1992; Solari and Pigozzi, 1993).

Genetic data show that crossingover between differentiated parts of the X and Y chromosomes is suppressed (Kirpichnikov, 1981). Non-homologous synapsis of the telomeric regions of the sex chromosomes, detected in our study, may be a mechanism of the suppression of crossingover.

P. sphenops x P. velifera hybrid

All stages of spermatogenesis (including mature sperm of normal morphology) were represented in testes of the male hybrids between these two species. The SC complement consisted of 23 bivalents, as expected . All bivalents demonstrated complete pairing at pachytene. No indication of heteromorphism for any chromosome was found. This implies that the divergence between P. sphenops and P. velifera was not accompanied by chromosomal rearrangements. The genic divergence did not lead to incompatibility of the species-specific genetic systems controlling meiotic progression and spermatogenesis.

P. velifera x P. reticulatus hybrid

One of two hybrid males was completely sterile: neither sperm, nor prophase spermatocytes were found in the spreads of its testes. A little sperm was found in the other male hybrid. Pachytene cells were abundant, but many of them showed definite signs of degeneration such as complete asynapsis of the axial elements and accumulation of large globules of electron dense material in the nuclei. A majority of pachytene cells demonstrated various pairing failures: univalents, interlocks, multiple non-homologous pairing and end-to-end associations. The number of chromosomes which failed to pair varied from cell to cell. In some cells, almost all chromosomes were unpaired (Figure 1a). In the other cells, some chromosomes were non-homologously paired with each other, forming chains, some displayed partial or complete asynapsis and some formed normal bivalents (Figure 1b). Figure 1g shows a cell in which all but three bivalents are completely normal. A few cells (three of 869) were found to contain only completely paired homomorphic bivalents (Figure 1c).

Fig 1a	Fig 1b	Fig 1c

The variable appearance of pairing disturbances and the finding normal spermatocytes (although extremely rare) imply that pairing failure in the majority of pachytene cells of the P. velifera x P. reticulatus hybrid, and arrest of spermatogenesis are determined by genic incompatibility of the species-specific mechanisms controlling meiotic prophase in the parental species, rather then loss of homology between their chromosomes. The finding of mature sperm (though a very small number) in the second hybrid male demonstrates that meiotic arrest in the hybrid is not complete, and some cells are able to surmount it.

Thus, we can conclude that divergence between P. reticulatus and P. velifera as well as between P. velifera and P. sphenops was not accompanied by gross chromosomal rearrangements. All of them have similar SC karyotypes. All of their chromosomes are able to form homomorphic bivalents in meiosis in the hybrid males. This confirms the conclusions about karyotypic similarity between these species inferred from comparative analysis of routinely stained karyotypes. However, the degree of genic divergence between these three species is substantially different. While no postzygotic reproductive barriers were formed between P. velifera and P. sphenops, the genetic system controlling meiotic prophase of the guppy has undergone significant divergence, and has become incompatible with those of mollies. In the latter case we are observing the final steps of formation of a postzygotic mechanism of isolation.

Ve denemenin sonucunda oluşan yavrular:

I picked up the aquarium hobby at a the fairly young age of 8--after spending most of the previous year researching and becoming increasingly more fascinated by the world of fish, "Santa"� left me a 10 gallon setup under the Christmas tree. I have a lot of very distinct memories of my own tanks and experiments, but only one extremely strong image from the literature which I devoured that year and is now in some good portion obsolete. I distinctly recall a slightly fuzzy black and white photograph of a very pretty semi-metallic mottled fish that was labeled as a "guppy x molly hybrid"�. That single fish fascinated me and stuck with me for many years. I tried to breed them myself in one of my experimental phases, stocking a 20-gallon community tank with just mollies and guppies in among the tetras, and of course met with no success, and soon moved on to other projects.

This past year, I began to build myself a proper fish room and really spend some time back in the hobby after some years of very casual, very minimal fish keeping. As I started running across some more unusual livebearers, starting with the Endler's livebearer and hetrandria formosa, and eventually moving on to halfbeaks and goodieds, I came back to the long-ago memory of the "guppy x molly hybrid"�. Some time on Google brought up the debate on message boards across the web, which I read all of, fascinated. It seems that even nearly 20 years since I first came across the creatures, they were still mostly a mystery and their existence still questionable. Most personal accounts of livebearer hybridization I saw were questionable at best--such as failing to account for the female's ability to store sperm for some months--and impossible at worst (balloon mollies are molly x platy hybrids, according to one dubious theory). So with much more to devote in terms of time and resources, I decided to work it out myself for once and for all.

I began with two female guppies--blonde with red tails--from a reputable local store who kept males and females in separate tanks, and whose wholesaler did the same. I picked the two that looked youngest, barely sexually mature, and with the body shape typical of a livebearer who has never carried a litter. I then isolated them in a 2-gallon tank, alone, for the next 4 months. Neither ever developed a gravid appearance or dropped any fry. A third female I raised myself, from a batch of 5 young fry I was given. The breeder net in the tank they were in tore before they were more than half an inch long, and 4 of the fry escaped into the tank and were eaten. This female, a silver half-black, was the sole survivor, and joined the other 2 girls in their isolation tank for the last month.

Surviving isolated gold female

black female (raised from fry)

After the females had remained under observation long enough to guarantee they were not retaining any sperm from previous matings with male guppies, I moved them to a 6-gallon Eclipse tank with many fake plants and a breeding trio of aoslene sphixi snails, and brought home two male mollies to join them. One male was a traditional, small, black molly, the second was a slightly larger “gold-dust� male. I selected smaller males that looked the most true to type as regards “traditional� mollies and avoided those that were obvious hybrids (within the molly family) with lyre tails or the much larger sail-fin influence.

Both males


The two groups of fish ignored each other for about a week and a half, then suddenly the males began courting the guppy girls in nearly a frenzy and both males were observed mating with all three females many times over several days.

Very fast, very blurry courtship!

And they held still for just the right split second!

Unfortunately, about a week later, one of the original blonde females died unexpectedly overnight, with no outvard signs of illness. This brought the ratio down to 2 of each gender (and species) and while the gold dust male continued to try to court the females, the smaller black male suddenly took to harassing the gold dust male and trying to mate with HIM so much the gold dust couldn’t get to the female guppies and quickly lost interest in attempting to mate at all. I removed the black male to an empty quarantine tank, but the gold dust had been so harassed he ignored the female guppies, so after two days I switched him out with the little black molly male, who instantly went after the girls again.

Roughly six weeks after the introduction, the remaining blonde female appeared extremely gravid and eyespots were visible through her abdomen. While the half-black female had filled out a little, she still lacked the traditional gravid appearance, so she and both males were removed to a healthy community tank of non-livebearers while the very gravid blonde female was left in the 6-gallon to deliver.

Five days later, October 7, 2006, the blonde female guppy--the only fish in her tank--dropped a small batch of fry. I was able to count at least four, and she still has some eyespots showing through her side, indicating she is likely not done with the birth. The fry are extremely large for a guppy, and have the body shape of a newborn molly, not a guppy. All the fry appear to be a muddy marbled/mottled color typical of mollies. If I had seen these fry in a tank with no knowledge of their heritage, I would have assumed they were simply slightly smaller than usual molly fry. At one day old, there is very little sign of the guppy parentage, although this may change as they age.

October 8, the blonde female was removed to a recovery tank. There are at least 6 fry, there is enough riccia, duckweed, and anacharis/elodea that an accurate count is difficult.

Photos of the fry at one day old:

Fry at 5 days old. The're eating crushed flake, frozen daphnia, and growing like weeds. Count is still, tenatively, 6.

They are now 24 days old

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